Products and methods for treatment of familial amyotrophic lateral sclerosis

ABSTRACT

The present invention relates to RNA-based methods for inhibiting the expression of the superoxide dismutase 1 (SOD-1) gene. Recombinant adeno-associated viruses of the invention deliver DNAs encoding RNAs that knock down the expression of SOD-1. The methods have application in the treatment of amyotrophic lateral sclerosis.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under U.S. NationalInstitutes of Health R21-NS067238, NS027036, ROI NS064492 and RC2NS69476-01. The Government has certain rights in the invention.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: 14,350 byte ACII (Text) file named“47886PCT_SeqListing.txt,” created on Aug. 26, 2014.

FIELD OF THE INVENTION

The present invention relates to RNA-based methods for inhibiting theexpression of the superoxide dismutase 1 (SOD-1) gene. Recombinantadeno-associated viruses of the invention deliver DNAs encoding RNAsthat knock down the expression of SOD-1. The methods have application inthe treatment of amyotrophic lateral sclerosis (ALS).

BACKGROUND

ALS is an adult-onset, rapidly progressive and fatal neurodegenerativedisease, characterized by selective degeneration of both upper and lowermotor neurons. First characterized by Charcot in 1869, ALS isresponsible for one in every 2000 deaths, affecting nearly 5 out of100,000 individuals. ALS occurs when specific nerve cells in the brainand spinal cord that control voluntary movement degenerate. Within twoto five years after clinical onset, the loss of these motor neuronsleads to progressive atrophy of skeletal muscles, which results in lossof muscular function resulting in paralysis, speech deficits, and deathdue to respiratory failure.

Most ALS cases have no clear genetic linkage and are referred to assporadic, but in 10% of instances disease is familial with dominantinheritance. Twenty percent of familial cases are caused by mutations inthe enzyme superoxide dismutase 1 (SOD1), with over 140 distinctmutations identified to date^(1,2). Many efforts to identify howmutations alter the function of SOD1 have produced a consensus view thatSOD1 mutants acquire one or more toxicities, whose nature still remainscontroversial³, but there is clear evidence that a proportion of mutantSOD1 is misfolded and subsequently aggregates^(4,5). SOD1 aggregatesare, in fact, one of the histological hallmarks of SOD1-related ALScases⁴.

In the past 20 years, multiple animal models expressing mutant forms ofhuman SOD1 have been generated. These models recapitulate the hallmarksof ALS, developing age-dependent motor axon degeneration andaccompanying muscle denervation, glial inflammation and subsequent motorneuron loss. Selective gene excision experiments have determined thatmutant SOD1 expression within motor neurons themselves contributes todisease onset and early disease progression⁶, as does mutant synthesisin NG2⁺ cells' that are precursors to oligodendrocytes. However, mutantSOD1 protein expression in microglia and astrocytes significantly drivesrapid disease progression^(6,8), findings which have lead to theconclusion that ALS pathophysiology is non-cell autonomous³.

Further, astrocytes have been found to be toxic to motor neurons inmultiple in vitro models where mutant forms of human SOD1 wereoverexpressed⁹⁻¹¹. A recent study derived astrocytes from post-mortemspinal cords of ALS patients with or without SOD1 mutations. In allcases, astrocytes from sporadic ALS patients were as toxic to motorneurons as astrocytes carrying genetic mutations in SOD1¹². Even morestrikingly, reduction of SOD1 in astrocytes derived from both sporadicand familial ALS patients decreased astrocyte-derived toxicity that isselective for motor, but not GABA, neurons. This remarkable finding,along with reports that misfolded SOD1 inclusions are found in thespinal cords of familial as well as some sporadic ALSpatients^(13,14,15), has provided strong evidence for a pathogenic roleof wild-type SOD1 in sporadic ALS.

Despite the insights that SOD1 mutant-expressing animal models haveprovided for understanding mechanisms involved in motor neurondegeneration, their utility for the development of therapeuticapproaches has been questioned¹⁶, as no drug with a reported survivalbenefit in mutant SOD1^(G93A) mice has been effective in clinical trialswith sporadic ALS patients. In all but one case the drugs taken to humantrial had been reported only to extend mutant SOD1 mouse survival whenapplied presymptomatically, and even then to provide a survival benefitsolely by delaying disease onset with no benefit in slowing diseaseprogression. The one exception to this was riluzole, which like thehuman situation, modestly extended survival of mutant SOD1^(G93A) miceand did so by slowing disease progression¹⁷. Recognizing that success athuman trial will require slowing of disease progression, the SOD1 mutantmice have perfectly predicted the success of riluzole and the failure ofefficacy of each other drug attempted in human trial. What has beenmissing are additional therapies that affect disease progression inthese mice.

Thus, riluzole is the only drug currently approved by the FDA as atherapy for ALS, providing a modest survival benefit²¹. For the 20% offamilial cases caused by mutation in SOD1, attempts at improving therapyby reducing synthesis of SOD1 have been the focus of multipletherapeutic development approaches. Antisense oligonucleotides and viraldelivered RNA interference (RNAi) were tested in rat²² and mousemodels²³⁻²⁵ that develop fatal paralysis from overexpressing humanSOD1^(G93A). Antisense oligonucleotides infused at disease onsetproduced SOD1 reduction and a modest slowing of disease progression²².Direct CSF infusion of antisense oligonucleotides has been testedclinically²⁶, leading to encouraging results in terms of tolerabilityand safety, but without significant reduction in SOD1 levels at the lowdosages utilized. In each of the prior viral studies²³⁻²⁵, SOD1knockdown was achieved before disease onset by direct injection into thenervous system or taking advantage of axonal retrograde transport when avirus was injected intramuscularly^(23, 24.) These studies led tovarying degrees of success in extending survival or improving motorperformance, depending on the time of treatment as well as level of SOD1knockdown achieved in the spinal cord. Although these studies providedimportant proof of principle, the approaches were far from being readilytranslated into clinical strategies. Indeed, there have beencontroversial reports surrounding these initial viral mediated SOD1suppression studies^(23, 24, 27-29).

Adeno-associated virus (AAV) vectors have been used in a number ofrecent clinical trials for treatment of neurological disorders [Kaplittet al., Lancet 369: 2097-2105 (2007); Marks et al., Lancet Neurol 7:400-408 (2008); Worgall et al., Hum Gene Ther (2008)].

AAV is a replication-deficient parvovirus, the single-stranded DNAgenome of which is about 4.7 kb in length including 145 nucleotideinverted terminal repeat (ITRs). The nucleotide sequence of the AAVserotype 2 (AAV2) genome is presented in Srivastava et al., J Virol, 45:555-564 (1983) as corrected by Ruffing et al., J Gen Virol, 75:3385-3392 (1994). Cis-acting sequences directing viral DNA replication(rep), encapsidation/packaging and host cell chromosome integration arecontained within the ITRs. Three AAV promoters (named p5, p19, and p40for their relative map locations) drive the expression of the two AAVinternal open reading frames encoding rep and cap genes. The two reppromoters (p5 and p19), coupled with the differential splicing of thesingle AAV intron (at nucleotides 2107 and 2227), result in theproduction of four rep proteins (rep 78, rep 68, rep 52, and rep 40)from the rep gene. Rep proteins possess multiple enzymatic propertiesthat are ultimately responsible for replicating the viral genome. Thecap gene is expressed from the p40 promoter and it encodes the threecapsid proteins VP1, VP2, and VP3. Alternative splicing andnon-consensus translational start sites are responsible for theproduction of the three related capsid proteins. A single consensuspolyadenylation site is located at map position 95 of the AAV genome.The life cycle and genetics of AAV are reviewed in Muzyczka, CurrentTopics in Microbiology and Immunology, 158: 97-129 (1992).

AAV possesses unique features that make it attractive as a vector fordelivering foreign DNA to cells, for example, in gene therapy. AAVinfection of cells in culture is noncytopathic, and natural infection ofhumans and other animals is silent and asymptomatic. Moreover, AAVinfects many mammalian cells allowing the possibility of targeting manydifferent tissues in vivo. Moreover, AAV transduces slowly dividing andnon-dividing cells, and can persist essentially for the lifetime ofthose cells as a transcriptionally active nuclear episome(extrachromosomal element). The AAV proviral genome is infectious ascloned DNA in plasmids which makes construction of recombinant genomesfeasible. Furthermore, because the signals directing AAV replication,genome encapsidation and integration are contained within the ITRs ofthe AAV genome, some or all of the internal approximately 4.3 kb of thegenome (encoding replication and structural capsid proteins, rep-cap)may be replaced with foreign DNA such as a gene cassette containing apromoter, a DNA of interest and a polyadenylation signal. The rep andcap proteins may be provided in trans. Another significant feature ofAAV is that it is an extremely stable and hearty virus. It easilywithstands the conditions used to inactivate adenovirus (56° to 65° C.for several hours), making cold preservation of AAV less critical. AAVmay even be lyophilized. Finally, AAV-infected cells are not resistantto superinfection.

Multiple serotypes of AAV exist and offer varied tissue tropism. Knownserotypes include, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,AAV7, AAV8, AAV9, AAV10, AAV11 and AAVrh74. Advances in the delivery ofAAV6 and AAV8 have made possible the transduction by these serotypes ofskeletal and cardiac muscle following simple systemic intravenous orintraperitoneal injections. See Pacak et al., Circ. Res., 99(4): 3-9(1006) and Wang et al., Nature Biotech., 23(3): 321-8 (2005). The use ofAAV to target cell types within the central nervous system has involvedsurgical intraparenchymal injection. See, Kaplitt et al., supra; Markset al., supra and Worgall et al., supra. Regarding the use of AAV totarget cell types within the nervous system, see InternationalPublication No. WO 2010/071832. International Publication Nos. WO2009/043936 and WO 2009/013290 state they relate to delivering genes tothe central nervous system. International Publication No. WO 2011/133890states it relates to recombinant adeno-associated viruses useful fortargeting transgenes to central nervous system tissue.

There thus remains a need in the art for methods and materials fortreatment of ALS.

SUMMARY

The present invention provides products and methods useful for reducingmutant SOD1 protein levels in subjects in need thereof. The inventionprovides AAV-mediated delivery of RNAs including, but not limited toshort hairpin RNAs, to reduce synthesis of ALS-causing human SOD1mutants in subjects in need thereof. Recombinant AAV (rAAV) contemplatedby the invention include, but are not limited to, rAAV9, rAAV2 andrAAVrh74. Delivery routes contemplated by the invention include, but arenot limited to, systemic delivery and intrathecal delivery. Use of themethods and products of the invention is indicated, for example, intreating ALS.

DETAILED DESCRIPTION

In one aspect, the invention provides rAAV genomes comprising one ormore AAV ITRs flanking a polynucleotide encoding one or more RNAs(including, but not limited to, small hairpin RNAs, antisense RNAsand/or microRNAs) that target mutant SOD1 polynucleotides. The examplesdescribe the use of exemplary rAAV encoding small hairpin RNAs (shRNAs).In the rAAV genomes, the shRNA-encoding polynucleotide is operativelylinked to transcriptional control DNA, specifically promoter DNA that isfunctional in target cells. Commercial providers such as Ambion Inc.(Austin, Tex.), Darmacon Inc. (Lafayette, Colo.), InvivoGen (San Diego,Calif.), and Molecular Research Laboratories, LLC (Herndon, Va.)generate custom inhibitory RNA molecules. In addition, commercially kitsare available to produce custom siRNA molecules, such as SILENCER™ siRNAConstruction Kit (Ambion Inc., Austin, Tex.) or psiRNA System(InvivoGen, San Diego, Calif.). In some embodiments, the rAAV genomecomprises a DNA encoding a SOD1 shRNA such as:

(SEQ ID NO: 1) GCATCATCAATTTCGAGCAGAAGGAA, (SEQ ID NO: 2)GAAGCATTAAAGGACTGACTGAA, (SEQ ID NO: 3) CTGACTGAAGGCCTGCATGGATT,(SEQ ID NO: 4) CATGGATTCCATGTTCATGA  (″shRNA 130″ or ″SOD1 shRNA″herein), (SEQ ID NO: 5) GCATGGATTCCATGTTCATGA, (SEQ ID NO: 6)GGTCTGGCCTATAAAGTAGTC, (SEQ ID NO: 7) GGGCATCATCAATTTCGAGCA,(SEQ ID NO: 8) GCATCATCAATTTCGAGCAGA, (SEQ ID NO: 9)GCCTGCATGGATTCCATGTTC, (SEQ ID NO: 10) GGAGGTCTGGCCTATAAAGTA,(SEQ ID NO: 11) GATTCCATGTTCATGAGTTTG, (SEQ ID NO: 12)GGAGATAATACAGCAGGCTGT, (SEQ ID NO: 13) GCTTTAAAGTACCTGTAGTGA,(SEQ ID NO: 14) GCATTAAAGGACTGACTGAAG, (SEQ ID NO: 1)GCATCATCAATTTCGAGCAGAAGGAA, (SEQ ID NO: 2) GAAGCATTAAAGGACTGACTGAA,(SEQ ID NO: 3) CTGACTGAAGGCCTGCATGGATT, (SEQ ID NO: 4)CATGGATTCCATGTTCATGA, (SEQ ID NO: 5) GCATGGATTCCATGTTCATGA,(SEQ ID NO: 6) GGTCTGGCCTATAAAGTAGTC, (SEQ ID NO: 7)GGGCATCATCAATTTCGAGCA, (SEQ ID NO: 8) GCATCATCAATTTCGAGCAGA,(SEQ ID NO: 9) GCCTGCATGGATTCCATGTTC, (SEQ ID NO: 10)GGAGGTCTGGCCTATAAAGTA, (SEQ ID NO: 11) GATTCCATGTTCATGAGTTTG,(SEQ ID NO: 12) GGAGATAATACAGCAGGCTGT, (SEQ ID NO: 13)GCTTTAAAGTACCTGTAGTGA, (SEQ ID NO: 14) GCATTAAAGGACTGACTGAAG,(SEQ ID NO: 15) TCATCAATTTCGAGCAGAA, (SEQ ID NO: 16)TCGAGCAGAAGGAAAGTAA, (SEQ ID NO: 17) GCCTGCATGGATTCCATGT,(SEQ ID NO: 18) TCACTCTCAGGAGACCATT,  or (SEQ ID NO: 19)GCTTTAAAGTACCTGTAGT.

The rAAV genomes of the invention lack AAV rep and cap DNA. AAV DNA inthe rAAV genomes (e.g., ITRs) may be from any AAV serotype for which arecombinant virus can be derived including, but not limited to, AAVserotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9,AAV-10 and AAV-11. The nucleotide sequences of the genomes of the AAVserotypes are known in the art. For example, the complete genome ofAAV-1 is provided in GenBank Accession No. NC_002077; the completegenome of AAV-2 is provided in GenBank Accession No. NC_001401 andSrivastava et al., J. Virol., 45: 555-564 {1983); the complete genome ofAAV-3 is provided in GenBank Accession No. NC_1829; the complete genomeof AAV-4 is provided in GenBank Accession No. NC_001829; the AAV-5genome is provided in GenBank Accession No. AF085716; the completegenome of AAV-6 is provided in GenBank Accession No. NC_00 1862; atleast portions of AAV-7 and AAV-8 genomes are provided in GenBankAccession Nos. AX753246 and AX753249, respectively; the AAV-9 genome isprovided in Gao et al., J. Virol., 78: 6381-6388 (2004); the AAV-10genome is provided in Mol. Ther., 13(1): 67-76 (2006); and the AAV-11genome is provided in Virology, 330(2): 375-383 (2004). The AAVrh74genome is provided in International Publication No. WO 2013/078316.

In another aspect, the invention provides DNA plasmids comprising rAAVgenomes of the invention. The DNA plasmids are transferred to cellspermissible for infection with a helper virus of AAV (e.g., adenovirus,E1-deleted adenovirus or herpesvirus) for assembly of the rAAV genomeinto infectious viral particles. Techniques to produce rAAV particles,in which an AAV genome to be packaged, rep and cap genes, and helpervirus functions are provided to a cell are standard in the art.Production of rAAV requires that the following components are presentwithin a single cell (denoted herein as a packaging cell): a rAAVgenome, AAV rep and cap genes separate from (i.e., not in) the rAAVgenome, and helper virus functions. The AAV rep and cap genes may befrom any AAV serotype for which recombinant virus can be derived and maybe from a different AAV serotype than the rAAV genome ITRs, including,but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5,AAV-6, AAV-7, AAV-8, AAV-9, AAV-10 and AAV-11. Production of pseudotypedrAAV is disclosed in, for example, WO 01/83692 which is incorporated byreference herein in its entirety. In various embodiments, AAV capsidproteins may be modified to enhance delivery of the recombinant vector.Modifications to capsid proteins are generally known in the art. See,for example, US 20050053922 and US 20090202490, the disclosures of whichare incorporated by reference herein in their entirety.

A method of generating a packaging cell is to create a cell line thatstably expresses all the necessary components for AAV particleproduction. For example, a plasmid (or multiple plasmids) comprising arAAV genome lacking AAV rep and cap genes, AAV rep and cap genesseparate from the rAAV genome, and a selectable marker, such as aneomycin resistance gene, are integrated into the genome of a cell. AAVgenomes have been introduced into bacterial plasmids by procedures suchas GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA,79:2077-2081), addition of synthetic linkers containing restrictionendonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73) orby direct, blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem.,259:4661-4666). The packaging cell line is then infected with a helpervirus such as adenovirus. The advantages of this method are that thecells are selectable and are suitable for large-scale production ofrAAV. Other examples of suitable methods employ adenovirus orbaculovirus rather than plasmids to introduce rAAV genomes and/or repand cap genes into packaging cells.

General principles of rAAV production are reviewed in, for example,Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka,1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Variousapproaches are described in Ratschin et al., Mol. Cell. Biol. 4:2072(1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984);Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J.Virol., 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol.,7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828); U.S. Pat.No. 5,173,414; WO 95/13365 and corresponding U.S. Pat. No. 5,658,776; WO95/13392; WO 96/17947; PCT/U598/18600; WO 97/09441 (PCT/US96/14423); WO97/08298 (PCT/US96/13872); WO 97/21825 (PCT/US96/20777); WO 97/06243(PCT/FR96/01064); WO 99/11764; Perrin et al. (1995) Vaccine13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark etal. (1996) Gene Therapy 3:1124-1132; U.S. Pat. No. 5,786,211; U.S. Pat.No. 5,871,982; and U.S. Pat. No. 6,258,595. Single-stranded rAAV arespecifically contemplated. The foregoing documents are herebyincorporated by reference in their entirety herein, with particularemphasis on those sections of the documents relating to rAAV production.

The invention thus provides packaging cells that produce infectiousrAAV. In one embodiment packaging cells may be stably transformed cancercells such as HeLa cells, 293 cells and PerC.6 cells (a cognate 293line). In another embodiment, packaging cells are cells that are nottransformed cancer cells such as low passage 293 cells (human fetalkidney cells transformed with E1 of adenovirus), MRC-5 cells (humanfetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells(monkey kidney cells) and FRhL-2 cells (rhesus fetal lung cells).

In still another aspect, the invention provides rAAV (i.e., infectiousencapsidated rAAV particles) comprising a rAAV genome of the invention.In some embodiments, the rAAV genome is a self-complementary genome. Thegenomes of the rAAV lack AAV rep and cap DNA, that is, there is no AAVrep or cap DNA between the ITRs of the genomes. Embodiments include, butare not limited to, the exemplary rAAV including a genome encoding theSOD1 shRNA named “AAV-SOD1-shRNA.” A sequence including theAAV-SOD1-shRNA genome is set out below as an inverted sequence from aplasmid used in production.

FEATURES Location/Qualifiers misc_feature 662..767 /gene=″mutated ITR″/SECDrawAs=″Region″ /SECStyleId=1 CDS complement (901..965)/gene=″SOD shRNA″ /SECDrawAs=″Gene″ /SECStyleId=1 misc_featurecomplement (966..1064) /gene=″H1″ /SECDrawAs=″Region″ /SECStyleId=1misc_feature 1224..1503 /gene=″CMV enhancer″ /SECDrawAs=″Region″/SECStyleId=1 misc_feature 1510..1779 /gene=″B-Actin promoter″/product=″Chicken″ /SECDrawAs=″Region″ /SECStyleId=1 misc_feature1845..1875 /gene=″SV40_late_19s_int″ /SECDrawAs=″Region″ /SECStyleId=1misc_feature 1845..1941 /gene=″modSV40_late_16s_int″ /SECDrawAs=″Region″/SECStyleId=1 CDS 2015..2734 /gene=″GFP″ /SECDrawAs=″Gene″ /SECStyleId=1misc_feature 2783..2929 /gene=″BGHpA″ /SECDrawAs=″Region″ /SECStyleId=1misc_feature 3009..3149 /gene=″ITR″ /SECDrawAs=″Region″ /SECStyleId=1misc_feature 3983..4843 /gene=″amp r″ /SECDrawAs=″Region″ /SECStyleId=1misc_feature 4997..5618 /gene=″pBR322 ori″ /SECDrawAs=″Region″/SECStyleId=1 (SEQ ID NO: 20)    1gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctgattct   61aacgaggaaa gcacgttata cgtgctcgtc aaagcaacca tagtacgcgc cctgtagcgg  121cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc  181cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc  241ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct  301cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac  361ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac  421tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat  481ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa  541aatattaacg cttacaattt aaatatttgc ttatacaatc ttcctgtttt tggggctttt  601ctgattatca accggggtac atatgattga catgctagtt ttacgattac cgttcatcgc  661cctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt  721tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggaat tcacgcgtgg  781atctgaattc aattcacgcg tggtacctac actttatgct tccggctcgt atgttgtgtg  841gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc  901tttccaaaaa agcatggatt ccatgttcat gatctcttga atcatgaaca tggaatccat  961ggatccgagt ggtctcatac agaacttata agattcccaa atccaaagac atttcacgtt 1021tatggtgatt tcccagaaca catagcgaca tgcaaatatg aattcactgg ccgtcgtttt 1081acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc 1141ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt 1201gcgcagcctg tggtacctct ggtcgttaca taacttacgg taaatggccc gcctggctga 1261ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca 1321atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca 1381gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg 1441cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc 1501tactcgaggc cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt 1561tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggggc 1621gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg 1681cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc 1741ggcggcccta taaaaagcga agcgcgcggc gggcgggagc gggatcagcc accgcggtgg 1801cggcctagag tcgacgagga actgaaaaac cagaaagtta actggtaagt ttagtctttt 1861tgtcttttat ttcaggtccc ggatccggtg gtggtgcaaa tcaaagaact gctcctcagt 1921ggatgttgcc tttacttcta ggcctgtacg gaagtgttac ttctgctcta aaagctgcgg 1981aattgtaccc gcggccgatc caccggtcgc caccatggtg agcaagggcg aggagctgtt 2041caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag 2101cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg 2161caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt 2221gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat 2281gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac 2341ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat 2401cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca 2461caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg 2521ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat 2581cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag 2641caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg 2701gatcactctc ggcatggacg agctgtacaa gtaaagcggc catcaagctt atcgataccg 2761tcgactagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt 2821tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa 2881taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg 2941gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggagaga 3001tcgatctgag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct 3061cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt 3121gagcgagcga gcgcgcagag agggagtggc cccccccccc ccccccccgg cgattctctt 3181gtttgctcca gactctcagg caatgacctg atagcctttg tagagacctc tcaaaaatag 3241ctaccctctc cggcatgaat ttatcagcta gaacggttga atatcatatt gatggtgatt 3301tgactgtctc cggcctttct cacccgtttg aatctttacc tacacattac tcaggcattg 3361catttaaaat atatgagggt tctaaaaatt tttatccttg cgttgaaata aaggcttctc 3421ccgcaaaagt attacagggt cataatgttt ttggtacaac cgatttagct ttatgctctg 3481aggctttatt gcttaatttt gctaattctt tgccttgcct gtatgattta ttggatgttg 3541gaatcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg 3601gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc 3661aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc 3721tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc 3781gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt 3841ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 3901tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 3961ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 4021ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga 4081tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa 4141gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct 4201gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat 4261acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga 4321tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc 4381caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat 4441gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa 4501cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac 4561tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa 4621agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc 4681tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc 4741ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag 4801acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta 4861ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa 4921gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc 4981gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat 5041ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga 5101gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt 5161ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata 5221cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac 5281cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg 5341ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg 5401tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag 5461cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct 5521ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc 5581aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt 5641ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg 5701tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga 5761gtcagtgagc gaggaagcgg aagagcThe SOD shRNA nucleotides 901-965 comprise the entire hairpin sequenceincluding the sense and antisense arms, stem loop and terminationsequence. The sequence in a forward orientation (with target sequencesagainst SOD1 underlined) is:

(SEQ ID NO: 21) 5′AATTCATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGACCACTCGGATCCATGGATTCCATGTTCATGATTCAAGAGATCATGAACATGGAATC CATGCTTTTTTGGAAA 3′

The rAAV of the invention may be purified by methods standard in the artsuch as by column chromatography or cesium chloride gradients. Methodsfor purifying rAAV vectors from helper virus are known in the art andinclude methods disclosed in, for example, Clark et al., Hum. GeneTher., 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med.,69: 427-443 (2002); U.S. Pat. No. 6,566,118 and WO 98/09657.

In another aspect, the invention contemplates compositions comprisingrAAV of the present invention. Compositions of the invention compriserAAV in a pharmaceutically acceptable carrier. The compositions may alsocomprise other ingredients such as diluents and adjuvants. Acceptablecarriers, diluents and adjuvants are nontoxic to recipients and arepreferably inert at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, or other organic acids; antioxidantssuch as ascorbic acid; low molecular weight polypeptides; proteins, suchas serum albumin, gelatin, or immunoglobulins; hydrophilic polymers suchas polyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween, pluronics or polyethylene glycol (PEG).

Titers of rAAV to be administered in methods of the invention will varydepending, for example, on the particular rAAV, the mode ofadministration, the treatment goal, the individual, and the cell type(s)being targeted, and may be determined by methods standard in the art.Titers of rAAV may range from about about 1×10², about 1×10³, about1×10⁴, about 1×10⁵, about 1×10⁶, about 1×10⁷, about 1×10⁸, about 1×10⁹,about 1×10¹⁰, about 1×10¹¹, about 1×10¹², about 1×10¹³ to about 1×10¹⁴or more DNase resistant particles (DRP) per ml. Dosages may also beexpressed in units of viral genomes (vg). Dosages may also vary based onthe timing of the administration to a human. These dosages of rAAV mayrange from about 1×10⁴, about 1×10⁵, about 1×10⁶, about 1×10⁷, about1×10⁸, about 1×10⁹, about 1×10¹⁰, about 1×10¹¹, about 1×10¹², about1×10¹³, about 1×10¹⁴, about 1×10¹⁵, about 1×10¹⁶ or more viral genomesper kilogram body weight in an adult. For a neonate, the dosages of rAAVmay range from about about 1×10⁴, about 3×10⁴, about 1×10⁵, about 3×10⁵,about 1×10⁶, about 3×10⁶, about 1×10⁷, about 3×10, about 1×10⁸, about3×10⁸, about 1×10⁹, about 3×10⁹, about 1×10¹⁰, about 3×10¹⁰, about1×10¹¹, about 3×10¹¹, about 1×10¹², about 3×10¹², about 1×10¹³, about3×10¹³, about 1×10¹⁴, about 3×10¹⁴, about 1×10¹⁵, about 3×10¹⁵, about1×10¹⁶, about 3×10¹⁶ or more viral genomes per kilogram body weight.

In another aspect, the invention contemplates compositions comprisingrAAV of the present invention. Compositions of the invention compriserAAV in a pharmaceutically acceptable carrier. The compositions may alsocomprise other ingredients such as diluents and adjuvants. Acceptablecarriers, diluents and adjuvants are nontoxic to recipients and arepreferably inert at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, or other organic acids; antioxidantssuch as ascorbic acid; low molecular weight polypeptides; proteins, suchas serum albumin, gelatin, or immunoglobulins; hydrophilic polymers suchas polyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween, pluronics or polyethylene glycol (PEG).

In still another aspect, the invention provides methods of transducing atarget cell with a rAAV of the invention, in vivo or in vitro. The invivo methods comprise the step of administering an effective dose, oreffective multiple doses, of a composition comprising a rAAV of theinvention to a subject, a subject (including a human being), in needthereof. If the dose is administered prior to onset/development of adisorder/disease, the administration is prophylactic. If the dose isadministered after the onset/development of a disorder/disease, theadministration is therapeutic. In embodiments of the invention, aneffective dose is a dose that alleviates (eliminates or reduces) atleast one symptom associated with the disorder/disease state beingtreated, that slows or prevents progression to a disorder/disease state,that slows or prevents progression of a disorder/disease state, thatdiminishes the extent of disease, that results in remission (partial ortotal) of disease, and/or that prolongs survival. An example of adisease contemplated for treatment with methods of the invention is ALS.“Treatment” according to the invention thus alleviates (eliminates orreduces) at least one symptom associated with the disorder/disease statebeing treated (for example, weight loss is eliminated or reduced by atleast 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% orgreater), that slows or prevents progression to (onset/development) of adisorder/disease state, that slows or prevents progression of adisorder/disease state, that diminishes the extent of disease, thatresults in remission (partial or total) of disease, and/or that prolongssurvival. In some embodiments, survival is prolonged by at least 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or greater.

Combination therapies are also contemplated by the invention.Combination as used herein includes both simultaneous treatment orsequential treatments. Combinations of methods of the invention withstandard medical treatments (e.g., riluzole) are specificallycontemplated, as are combinations with novel therapies.

Administration of an effective dose of the compositions may be by routesstandard in the art including, but not limited to, systemicintramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary,intracranial, intrathecal, intraosseous, intraocular, rectal, orvaginal. Route(s) of administration and serotype(s) of AAV components ofthe rAAV (in particular, the AAV ITRs and capsid protein) of theinvention may be chosen and/or matched by those skilled in the arttaking into account the infection and/or disease state being treated andthe target cells/tissue(s) that are to express the SOD1 shRNAs. In someembodiments, the route of administration is systemic. In some,embodiments the route of administration is intrathecal. In some,embodiments the route of administration is introcerebroventricular. Insome, embodiments the route of administration is cisterna magna. Insome, embodiments the route of administration is by lumbar puncture.

Transduction of cells with rAAV of the invention results in sustainedexpression of SOD1 shRNAs. In another aspect, the present invention thusprovides methods of administering/delivering rAAV which express SOD1shRNA to a subject, preferably a human being. The term “transduction” isused to refer to the administration/delivery of SOD1 shRNAs to arecipient cell either in vivo or in vitro, via a replication-deficientrAAV of the invention resulting in expression of a SOD1 shRNA by therecipient cell.

Thus, the invention provides methods of administering an effective dose(or doses, administered essentially simultaneously or doses given atintervals) of rAAV that encode SOD1 shRNAs to a subject in need thereof.

In one aspect, the invention provides methods of delivering apolynucleotide encoding an shRNA of the invention across the BBBcomprising systemically administering a rAAV with a genome including thepolynucleotide to a subject. In some embodiments, the rAAV genome is aself complementary genome. In other embodiments, the rAAV genome is asingle-stranded genome. In some embodiments, the rAAV is a rAAV9. Insome embodiments, the rAAV is a rAAV2. In some embodiments, the rAAV isa rAAVrh74.

In some embodiments, the methods systemically deliver polynucleotidesacross the BBB to the central and/or peripheral nervous system.Accordingly, a method is provided of delivering a polynucleotide to thecentral nervous system comprising systemically administering a rAAV witha self-complementary genome including the genome to a subject. In someembodiments, the polynucleotide is delivered to brain. In someembodiments, the polynucleotide is delivered to the spinal cord. Alsoprovided is a method of delivering a polynucleotide to the peripheralnervous system comprising systemically administering a rAAV with aself-complementary genome including the polynucleotide to a subject isprovided. In some embodiments, the polynucleotide is delivered to alower motor neuron. In some embodiments, the rAAV genome is a selfcomplementary genome. In other embodiments, the rAAV genome is asingle-stranded genome. In some embodiments, the rAAV is a rAAV9. Insome embodiments, the rAAV is a rAAV2. In some embodiments, the rAAV isa rAAVrh74.

In another aspect, the invention provides methods of delivering apolynucleotide to the central nervous system of a subject in needthereof comprising intrathecal delivery of rAAV with a genome includingthe polynucleotide. In some embodiments, the rAAV genome is a selfcomplementary genome. In other embodiments, the rAAV genome is asingle-stranded genome. In some embodiments, the rAAV is a rAAV9. Insome embodiments, the rAAV is a rAAV2. In some embodiments, the rAAV isa rAAVrh74. In some embodiments, a non-ionic, low-osmolar contrast agentis also delivered to the subject, for example, iobitridol, iohexol,iomeprol, iopamidol, iopentol, iopromide, ioversol or ioxilan.

Embodiments of the invention employ rAAV to deliver polynucleotides tonerve, glial cells and endothelial cells. In some embodiments, the nervecell is a lower motor neuron and/or an upper motor neuron. In someembodiments, the glial cell is a microglial cell, an oligodendrocyteand/or an astrocyte. In other aspects the rAAV is used to deliver apolynucleotide to a Schwann cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. AAV9 transduction pattern and persistence in SOD1^(G93A) mice.SOD1^(G93A) mice were injected intravenously with AAV9-CB-GFP at P1, P21and euthanized 21 days post injection (n=3 per time point). Spinal cordswere examined for GFP, ChAT (motor neuron marker) and GFAP (astrocytemarker) expression. Temporal vein injection of AAV9-CB-GFP at P1resulted in efficient transduction of motor neurons and glia inSOD1^(G93A) mice (a,f,k,p). Tail vein injection at P21 (b,g,l,q)predominantly targeted astrocytes with few GFP positive motor neurons.To test the persistence of transduced cells, AAV9-CB-GFP wasintravenously injected at P1 and P21 in SOD1^(G93A) animals that weresacrificed at end stage (˜P130). Immunofluorescence analysis of lumbarventral horn (c,d,h,i,m,n,r,s) demonstrated that GFP expression wasmaintained in astrocytes throughout the disease course. To determinewhether SOD1 mediated inflammation and damage would affect AAV9transduction, we intravenously injected SOD1^(G93A) mice at P85 andharvested their spinal cords at endstage. There was no differenceobserved in the transduction pattern of SOD1^(G93A) mice treated at P21or P85. Insets in (r-t) show co-localization between GFP and GFAPsignal. (u) Quantification of transduced cells in ALS spinal cords (foreach group tissues were analyzed from 3 animals). GFP and ChAT columnsshow numbers of cells counted. Bars=100 μm. AAV, adeno-associated virus;P1, postnatal day 1; P21, postnatal day 21; P85, postnatal day 85; GFP,green fluorescent protein; ChAT, choline acetyltransferase; GFAP, glialfibrillary acidic protein.

FIG. 2. shRNA constructs show efficient reduction of human SOD1 proteinin vitro and in vivo. (a) Sequence alignments between human and mouseSOD1 for the regions targeted by the 4 different shRNA constructstested. (b) shRNA sequences were cloned into an H1 expression constructand transiently transfected into 293 cells. Lysates were collected 72hours post transfection and analyzed by western blot. (c) Quantificationof in vitro suppression of human SOD1 from three separate transienttransfections showed >50% reduction in SOD1. (d) shRNA 130 was packagedinto AAV9 and injected into SOD1^(G93A) mice at either P1 or P21. Spinalcords (n=3 per time point) were harvested three weeks post injection andanalyzed by western blot for human SOD1 protein levels. (e)Quantification of in vivo suppression of human SOD1 within the spinalcord of ALS mice. P1 and P21 injected spinal cords showed 60% and 45%reductions in mutant SOD1 protein, respectively. hSOD1, human superoxidedismutase 1; mSOD1, mouse superoxide dismutase 1; GAPDH, glyceraldehyde3 phosphate dehydrogenase.

FIG. 3. Intravenous delivery of AAV9-SOD1-shRNA improves survival andmotor performance in SOD1^(G93A) mice. SOD1^(G93A) mice received asingle intravenous injection of AAV9-SOD1-shRNA at P1 (n=6, green), P21(n=9, red) or P85 (n=5, blue). Treated mice were monitored up to endstage and compared with non-injected control SOD1^(G93A) mice (n=15,gray). (a,c) AAV9-SOD1-shRNA injection into P1 SOD1^(G93A) micesignificantly delayed median disease onset 39.5 days compared to controlanimals (uninjected, 103d; P1, 142.5d; p<0.05). Injection in P21 (red)or P85 (blue) ALS animals had no effect on disease onset (P21, 110d;P85, 105d). However, AAV9-SOD1-shRNA administered at P1, P21 or P85 allsignificantly extended median survival (b,e) (uninjected, 132d; P1,183.5d P21, 171d; P85, 162d; all comparisons to control p<0.001). TheP21 group had a significant extension in median disease duration (d)indicating a slowing of disease (uninjected, 29.5d; P1, 41d; P21, 49d;P85, 40d; Wilcoxon Signed Rank Test p=0.06, 0.01 and 0.12,respectively). (f-h) P1 and P21 treated animals maintained theirweights, had better hind limb grip strength and rotarod performance whencompared to age-matched controls, indicating treated animals retainedmuscle tone and motor function during their prolonged survival. Linesbetween bars in (c-e) indicate statistically significant differences. *p<0.05. P1, postnatal day 1; P21, postnatal day 21; P85, postnatal day85.

FIG. 4. Intravenous injection of AAV9-SOD1-shRNA reduces mutant proteinin spinal cords of SOD1^(G93A) mice. (a-d) Images of lumbar spinal cordsections from uninjected (a), P1 injected (b), P21 injected (c) and P85injected (d) mice were captured with identical microscope settings toqualitatively show SOD1 levels at end stage. SOD1 levels inverselycorrelate with survival. (e-t) Co-labeling for GFP, ChAT and SOD1 showsthat AAV9 transduced motor neurons had reduced SOD1 expression (arrows)while cells that lacked GFP maintained high levels of mutant protein(arrowheads). As described in FIG. 1u , higher MN transduction andcorresponding SOD1 reduction was observed in P1 injected mice (i-l) ascompared to P21 injected (m-p) and P85 injected (q-t) mice. Bar=100 μm.P1, postnatal day 1; P21, postnatal day 21; P85, postnatal day 85; SOD1,superoxide dismutase 1; GFP, green fluorescent protein; ChAT, cholineacetyltransferase.

FIG. 5. AAV9-SOD1-shRNA improves survival and motor performance inSOD1^(G37R) mice treated after disease onset. (a) There was nodifference in median disease onset between AAV9-SOD1-shRNA and controltreated mice. (average age at treatment=215d versus median onset of 194dcontrol and 197d treated; Log Rank Test p=0.46). (b,f) Median survivalof AAV9-SOD1-shRNA treated SOD1^(G37R) mice (n=25) was significantlyextended versus control mice (n=21). (control, n=21, 392d; SOD1 shRNA,n=25, 478.5d; Log Rank Test p<0.0001) (c-e) The early phase of diseasewas significantly slowed by 73 days in treated mice as compared tocontrol mice (control, 89d; SOD1 shRNA, 162d; p<0.0001 Wilcoxon SignedRank Test) while the late phase of disease showed a non-significantslowing (control, 63d; SOD1 shRNA, 81d; p=0.14 Wilcoxon Signed RankTest). Together this amounted to a 66 day increase in median diseaseduration (control, 173d; SOD1 shRNA, 239d; p<0.0001 Wilcoxon Signed RankTest). (g) A trend to improved hind limb grip strength appeared inAAV9-SOD1-shRNA treated mice compared to control mice.

FIG. 6. Intravenous injection of AAV9 in adult SOD1^(G37R) mice targetsastrocytes and motor neurons within the spinal cord. (a-h)Immunofluorescence analysis revealed neuronal as well as glialtransduction in both AAV9-CB-GFP (a-d) and AAV9-SOD1-shRNA treated (e-h)mice. (i-p) Human SOD1 levels appeared reduced in AAV9-SOD1-shRNAtreated mice (o) compared with AAV9-GFP treated mice (k). Bar=100 μm.GFP, green fluorescent protein; ChAT, choline acetyltransferase; GFAP,glial fibrillary acidic protein; SOD1, superoxide dismutase 1.

FIG. 7. Intrathecal infusion of AAV9-SOD1-shRNA in non-human primatesleads to efficient reduction in SOD1 levels. (a) A myelogram shortlyafter intrathecal infusion of AAV9-SOD1-shRNA mixed with contrast showsproper delivery into the subarachnoid space of a cynomolgus macaque.Arrows show diffusion of the contrast agent along the entire spinalcord. (b) Lumbar spinal cord sections from treated monkeys (n=3) wereharvested two weeks post injection and stained for GFP using DABstaining. Sections had widespread GFP expression throughout the grey andwhite matter. (c-e) Immunofluorescence analysis of the lumbar spinalcord sections showed robust GFP (c) expression within ChAT (d) positivecells indicating motor neuron transduction (e, merge). (f) Western blotanalysis of the lumbar spinal cords showed significant reduction in SOD1levels in AAV9-SOD1-shRNA injected animals as compared to controls. (g)In vivo quantification of SOD1 knockdown in monkey lumbar spinal cordhomogenate (n=3) showed an 87% reduction in animals that receivedAAV9-SOD1-shRNA compared to uninjected controls. (h) Laser capturemicrodissection was used to collect motor neurons or surroundingneuropil from injected and control lumbar monkey sections. Collectedcells were analyzed for SOD1 levels by qRT-PCR. Motor neurons collectedfrom AAV9-SOD1-shRNA animals (n=3) had a 95±3% reduction in SOD1 RNA.Non-neurons had a 66±9% reduction in SOD1 RNA in AAV9-SOD1-shRNA treatedanimals. Scale Bars: b=100 μm; e=50 μm. SOD1: Superoxide dismutase 1.

FIG. 8. Lumbar intrathecal infusion of AAV9-SOD1-shRNA leads toefficient transduction of motor neurons and non-neuronal cells in thecervical, thoracic and lumbar cord resulting in reduction of SOD1. (a-c)Immunofluorescence analysis of the three segments of the spinal cord;cervical (a), thoracic (b) and lumbar (c), showed robust GFP (green)expression within Chat (red) positive cells indicating motor neurontransduction. (d) GFP+/Chat+ cell counts show a caudal to rostralgradient of motor neuron transduction ranging from 85% of transducedcells in the lumbar region to more than 50% in the cervical region. (e)SOD1 mRNA levels in cervical, thoracic and lumbar cord sectionhomogenates analyzed by qRT-PCR show significant reduction in SOD1transcript, consistently with motor neuron transduction. SOD1 levelswere normalized to β-actin and AAV9-SOD1-shRNA injected animals werecompared to an AAV9-CB-GFP injected control. Scale bars: (a-c)=50 μm;Error bars: (d-e)=SD.

FIG. 9. Design of a clinical SOD1 shRNA construct. (a) Original AAV SOD1shRNA construct contains shRNA sequence against human SOD1 under H1promoter followed by the expression cassette for GFP which includes CMVenhancer, CBA promoter, modified SV40 intron, and GFP transgene sequencefollowed by bGH PolyA terminator. SOD1 shRNA expression cassette and GFPexpression cassette are flanked by AAV2 ITRs which ensures the packagingof the complete flanked sequence in AAV9 capsid. (b) In clinical SOD1shRNA construct, the GFP expression cassette is replaced by a stufferelement that contains tandem, noncoding sequences from FDA approved DNAvaccines. ITR: inverted terminal repeats; shRNA, small hairpin RNA;SOD1, superoxide dismutase 1; CMV, cytomegalo virus enhancer; CBA,Chicken β-actin promoter; GFP, green fluorescent protein; bGH pA, bovinegrowth hormone poly A terminator.

FIG. 10. Schematic of clinical SOD1 shRNA construct. Differentrestriction sites are placed in the clinical SOD1 shRNA construct thatallow the cloning of multiple shRNA expression cassettes whilemaintaining the total distance between the two ITRs.

FIG. 11. In vitro transfection of clinical SOD1 shRNA constructefficiently reduces human SOD1 protein in HEK293 cells. Representativemicroscopic fields showing bright-field images of non-transfectedcontrol (a), AAV SOD1 shRNA transfected (b) and shuttle vector pJet SOD1shRNA transfected (c,d) HEK 293 cells, 72 hrs post transfection.Corresponding fluorescence images reveal the lack of GFP fluorescencefrom pJet SOD1 shRNA transfected HEK 293 cells (g,h) as compared to AAVSOD1 shRNA transfected cells (f). (i) Western blot analysis of the celllysates confirms the efficient knockdown of human SOD1 protein in pJetSOD1 shRNA transfected cells as compared to the non-transfected controlcells. Immunoblot analysis also confirms removal of GFP transgene frompJet SOD1 shRNA construct. (j) Quantification of the in vitrodownregulation of SOD1 by pJet SOD1 shRNA. pJet SOD1 shRNA reduces theprotein levels of human SOD1 by almost 50% in HEK293 cells as comparedto control. This reduction is similar to that achieved with AAV SOD1shRNA construct.

FIG. 12. Schematic of cloning strategy for clinical AAV SOD1 shRNAvector. Clinical SOD1 shRNA construct was cloned into AAV CB MCS vectorusing Kpn1/SPh1 sites. Kpn1/SPh1 double digest of AAV CB MCS plasmidresults in the release of the complete transgene expression cassettefrom this vector which is further replaced with clinical SOD1 shRNAconstruct carrying SOD1 shRNA expression cassette and stuffer sequence.

FIG. 13. Clinical AAV SOD1 shRNA efficiently reduces human SOD1 levelsin vitro. HEK293 cells were transfected with clinical AAV SOD1 shRNAplasmid by Calcium phosphate method. Representative microscopic fieldsshowing brightfield images of non-transfected control, AAV SOD1 shRNAand Clinical AAV SOD1 shRNA transfected cells respectively, 72 hrspost-transfection (a-c). Successful removal of GFP from clinical AAVSOD1 shRNA was confirmed by lack of GFP expression in Clinical AAV SOD1shRNA transfected cells (f,g). (g) Western blot analysis of celllysates, harvested 72 hrs post-tranfection confirmed efficientdownregulation of SOD1 in clinical AAV SOD1 shRNA transfected cells ascompared to control. AAV SOD1 shRNA was used as a positive control. (h)Quantification of the in vitro knockdown of SOD1 by clinical AAV SOD1shRNA.

Figure S1. AAV9-shRNA-SOD1 administration is well tolerated in WT mice.Female and male WT animals were injected with AAV9-SOD1-shRNA at P1 orP21 and monitored up to 6 months of age. (a,b) Both male and femaletreated mice showed steady increase in body mass as compared to controlanimals. (c,d) Rotarod performance and (e,f) hind limb grip strengthwere not affected by P1 or P21 treatment in both groups as compared torespective controls. n=5 per group. WT, wild type; P1, postnatal day 1;P21, postnatal day 21.

Figure S2. Hematology and Serum Chemistry of AAV9-SOD1-shRNA treated WTanimals. (a-m) Blood was collected from P1 (green) or P21 (red) treatedand control (gray) WT animals at 150 days of age for hematology studies.No significant differences were observed between treated and controlanimals. (n-w) Serum samples collected at 180 days of age from the samemice showed no significant differences in serum chemistry profile.Mean±SEM. n=5 per group. P1, postnatal day 1; P21, postnatal day 21.

Figure S3. AAV9-SOD1-shRNA treatment in SOD1^(G93A) mice reducesastrogliosis. End stage sections from control and AAV9-SOD1-shRNAtreated animals were harvested and stained for GFAP, an astrocyteactivation marker. P1 (b) and P85 (d) injected mice showed reducedlevels of astrogliosis as compared to control (a) mice while P21(c)injected mice showed the maximum reduction. This was consistent with thepercent astrocyte transduction achieved in these mice (FIG. 1u ).However, no effect was observed on microglia reactivity (e-h). Bar=100μm. P1, postnatal day 1; P21, postnatal day 21; P85, postnatal day 85.

Figure S4. Intravenous injection of AAV9-SOD1-shRNA efficiently reduceslevels of mutant SOD1 protein in spinal cords of SOD1^(G37R) mice. (a)Following disease onset, AAV9-CB-GFP or AAV9-SOD1-shRNA was injected inSOD1^(G37R) mice and spinal cords were harvested at end stage andanalyzed by western blot for human SOD1 protein levels. (b)Quantification of a) shows suppression of human SOD1 within the spinalcord of SOD1^(G37R) mice (n=4 per group). hSOD1, human superoxidedismutase 1; GAPDH, glyceraldehyde 3 phosphate dehydrogenase.

Figure S5. shRNA 130 efficiently reduces the levels of monkey SOD1 invitro. (a) Sequence alignment of the region targeted by SOD1 shRNA 130and a single mismatch with the monkey sequence. Monkey sequencecorresponds to SOD1 sequence from Rhesus monkey (NM 001032804.1),Cynomolgus monkey (sequenced in-house) and African green monkey. (b) TheshRNA 130 expression cassette was cloned into lentiviral vector and usedto infect Cos-7 cells. Lysates were analyzed 72 hours post infection byqRT PCR for SOD1. shRNA 130 reduced SOD1 transcript levels by 75% inCos-7 cells.

EXAMPLES

The present invention is illustrated by the following examples. Whilethe present invention has been described in terms of various embodimentsand examples, it is understood that variations and improvements willoccur to those skilled in the art. Therefore, only such limitations asappear in the claims should be placed on the invention.

Example 1 AAV9 Transduction Pattern and Persistence in SOD1^(G93A) Mice

We first evaluated the efficiency of AAV9 transduction in theSOD1^(G93A) mouse model that develops fatal paralytic disease. High copySOD1^(G93A) mice were obtained from Jackson Laboratories (Bar Harbor,Me.) and bred within the Kaspar lab. Animals were genotyped before thetreatment to obtain SOD1^(G93A) expressing mice and their wild typelittermates. Only female mice were included in the SOD1^(G93A)experiments. Animals were injected intravenously at postnatal day 1 orday 21 (to be referred to as P1 and P21, respectively) withself-complementary AAV9 expressing GFP from the CMV enhancer/beta-actin(CB) promoter (AAV9-CB-GFP) (n=3 per group). Three weeks post-injection,animals were sacrificed, and spinal cords examined for GFP expression(FIGS. 1a-u ).

All procedures with animals described herein were performed inaccordance with the NIH Guidelines and approved by the ResearchInstitute at Nationwide Children's Hospital (Columbus, Ohio), Universityof California (San Diego, Calif.) or Mannheimer Foundation (Homestead,Fla.) Institutional Animal Care and Use Committees.

Transduction efficiency was high in SOD1^(G93A) astrocytes with GFPexpressed in 34±2% and 54±3%, respectively, of P1 and P21 injectedspinal grey matter astrocytes (defined by immunoreactivity for GFAP).This efficiency was similar to our previous report of 64±1% in P21injected wild type animals¹⁸. Motor neurons were a prominent cell typetransduced at all levels of the spinal cords of P1 injected SOD1^(G93A)animals (62±1%), compared with significantly lower targeting to motorneurons in P21 injected animals (8±1%).

Although we have previously reported that transduced astrocytes in wildtype spinal cords persist with continued GFP accumulation for at least 7weeks post injection¹⁸, longevity of mutant SOD1 astrocytes (and theircontinued synthesis of genes encoded by the AAV9 episome) during activeALS-like disease was untested. Therefore, SOD1^(G93A) mice were injectedat P1 and P21 with AAV9-CB-GFP and followed to end-stage (˜P130, n=3 pergroup) (FIGS. 1 c,d,h,i,m,n,r,s). Immunofluorescent examination of theend-stage SOD1^(G93A) spinal cords from animals injected at P1 and P21showed a comparable number of GFP-expressing astrocytes as were found 21days after AAV9 injection (P1: 42±2%, P21: 61±2%). These data areconsistent with survival of transduced astrocytes for the duration ofdisease (˜110 days post injection at P21) in SOD1^(G93A) mice and thatAAV9-encoded gene expression is maintained.

Further, recognizing that SOD1 mutant mediated damage, includingastrocytic and microglial activation and early changes in the bloodbrain barrier develop during disease in mice in SOD1 mutant mice²⁰, wetested if this damage affected AAV9 transduction. SOD1^(G93A) mice wereinjected at P85 with AAV9-CB-GFP and sacrificed at endstage (n=3) (FIGS.1 e,j,o,t). Analysis of the spinal cords revealed that the transductionpattern seen in P85 animals was similar to P21 treated animals withastrocytes as the predominant cell type transduced at all levels (51±6%GFP+/GFAP+ cells in lumbar grey matter).

Example 2 Development of an shRNA Sequence Specific for Human SOD1

To specifically target the human SOD1 mRNA, four shRNA constructstargeting human SOD1 were generated and obtained from the LifeTechnologies design tool. The constructs that had a minimum of four basemismatches compared to the mouse mRNA sequence (FIG. 2a ). The basenumbers for the human sequences shown correspond to record numberCCDS33536.1 in the NCBI CCDS database. These constructs were cloned inpSilencer 3.1 (Genscript) under the human H1 promoter and tested invitro. shRNA 130 along with H1 promoter was further cloned into an AAVvector along with a reporter GFP under Chicken Beta-Actin promoter toidentify the transduced cells. Human 293 cells were transfected witheach cassette. The HEK-293 cells were maintained in IMDM mediumcontaining 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin. Uponreaching ˜60% confluence, cells were transfected with pSilencer 3.1containing the shRNAs being tested. Protein lysates were prepared 72hours post transfection and analyzed for SOD1 levels by western blot.All four sequences reduced SOD1 protein levels by >50% (FIGS. 2b,c ).

shRNA130 was selected for further experiments because it produced themost consistent knockdown across three separate transfectionexperiments. It was cloned into a self-complementary AAV9 vector thatalso contained a GFP gene whose expression would identify transducedcells (referred to as AAV9-SOD1-shRNA). Self-complementaryAAV9-SOD1-shRNA was produced by transient transfection procedures usinga double-stranded AAV2-ITR-based CB-GFP vector, with a plasmid encodingRep2Cap9 sequence as previously described along with an adenoviralhelper plasmid pHelper (Stratagene, Santa Clara, Calif.) in 293 cells¹⁸.

To confirm that the shRNA could suppress accumulation of human SOD1,SOD1^(G93A) mice (n=3) were injected intravenously with AAV9-SOD1-shRNAat either P1 or P21. For neonatal mouse injections, postnatal day 1-2SOD1^(G93A) pups were utilized. Total volume of 50 μl containing 5×10¹¹DNAse resistant viral particles of AAV9-SOD1-shRNA (Virapur LLC, SanDiego, Calif.) was injected via temporal vein as previously described¹⁸.A correct injection was verified by noting blanching of the vein. Afterthe injection, pups were returned to their cage. Animals were euthanizedthree weeks post injection and the spinal cords were harvested andanalyzed by immunoblotting for both human (mutant) and murine(wild-type) SOD1 protein. P1 and P21 injected spinal cords showed 60%and 45% reductions in mutant SOD1 protein, respectively (FIGS. 2d,e ).Murine SOD1 levels remained unchanged in response to human SOD1knockdown.

Example 3 AAV9-SOD1-shRNA is Safe and Well Tolerated in Wild Type Mice

To determine whether high dose AAV9-SOD1-shRNA would be safe, normalmice of both sexes were intravenously injected at P1 or P21 (P1=5 males,5 females at 5×10¹¹vg; P21=5 males, 5 females at 2×10¹² vector genomes(vg)) and then monitored up to 6 months of age. Both P1 and P21 injectedmice showed a steady increase in body mass similar to untreated mice(Figure S1). Weekly behavioral tests observed no significant differencesbetween injected and control groups in motor skills (measured byrotarod) as well as in hind limb grip strength. At 150 and 180 days ofage, blood samples were collected. Complete and differential bloodcounts of both treated and untreated groups showed similar bloodchemistry parameters (Figure S2). Serum samples from both groups showedno significant differences in the levels of alkaline phosphatase,creatinine, blood urea nitrogen, potassium, sodium and chloride.Finally, all the animals were sacrificed at the age of 180 days.Histopathological analyses by a pathologist blinded to treatment grouprevealed no significant alterations in the AAV9-SOD1-shRNA treatedanimals compared to uninjected controls (data not shown). We concludethat both administration of AAV9 and sustained shRNA expression wereapparently safe and well tolerated.

Example 4 Extended Survival of SOD1^(G93A) Mice from AAV9 MediatedReduction in Mutant SOD1 Even when Initiated Mid-Disease

To test the efficacy of AAV9-mediated SOD1 reduction, we treated cohortsof SOD1^(G93A) mice with a single intravenous injection ofAAV9-SOD1-shRNA before (P1, 5×10¹¹vg, n=6 and P21, 2×10¹² vg, n=9) orafter (P85, 3×10¹² vg, n=5) onset, recognizing that many astrocytes, butfew motor neurons, would be transduced at the two later time points. Foradult tail vein injections, animals were placed in a restraint thatpositioned the mouse tail in a lighted, heated groove. The tail wasswabbed with alcohol then injected intravenously with AAV9-SOD1-shRNA.

Onset of disease (measured by weight loss from denervation-inducedmuscle atrophy) was significantly delayed by a median of 39.5 days (FIG.3a,c ; uninjected, 103d; P1, 142.5d; p<0.05, Wilcoxon Signed Rank Test)in the P1 injected cohort, but was not affected by either of the laterinjections (P21, 110d; P85, 105d). P1 and P21 treated animals maintainedtheir weights, had better rotarod performance and hind limb gripstrength when compared to age-matched controls, indicating treatedanimals maintained muscle tone and motor function during their prolongedsurvival (FIGS. 3f-h ). Survival was significantly extended by AAV9injection at all three ages, yielding survival times 30-51.5 days beyondthat of uninjected SOD1^(G93A) mice (uninjected, 132d; P1, 183.5d; P21,171d; P85, 162d; Log-Rank Test p=<0.0001, 0.0003 and 0.001,respectively) (FIGS. 3b,e ). Defining disease duration as the time fromonset to end-stage revealed that the P21 treatment group hadsignificantly increased duration, indicative of slowed diseaseprogression, compared to uninjected controls (uninjected, 29.5d; P21,49d; Wilcoxon Signed Rank Test p=0.01), with trends toward slowedprogression in animals injected at the other two ages (P1, 41d; P85,40d; p=0.06 and 0.12, respectively) (FIG. 3d ). The lower percentage oftargeted non-neuronal cells at P1 versus those targeted at P21 (FIG. 1u) suggests that a minimum number of non-neuronal cells must be targetedto slow disease progression in the fast progressive SOD1^(G93A) model(FIG. 1u ).

Example 5 Reduction of Mutant SOD1 in AAV9 Infected Cells in TreatedSOD1^(G93A) Mice

Indirect immunofluorescence with an antibody that recognizes human, butnot mouse SOD1, was used to determine accumulated mutant SOD1 levels inend-stage spinal cords of treated and control mice. Human SOD1 levels inend-stage spinal cord sections inversely correlated with increasedsurvival (FIGS. 4a-d ). At end-stage, P1 (FIG. 4b ), P21 (FIG. 4c ) andP85 (FIG. 4d ) AAV9-SOD1-shRNA injected animals had lower levels ofmutant SOD1 when compared with uninjected SOD1^(G93A) animals (FIG. 4a). SOD1 expression within transduced motor neurons (identified by GFPand ChAT expressing cells) was reduced compared to surrounding neuronsthat had not been transduced to express viral encoded GFP (FIGS. 4h,l,p,t; arrows versus arrowheads). Moreover, immunofluorescence imagingof end-stage spinal cords revealed corresponding reduction inastrogliosis, but no difference in microgliosis in AAV9-SOD1-shRNAtreated animals versus controls (Figure S3).

Example 6 Therapeutic Slowing of Disease Progression with PeripheralInjection of AAV9 after Onset

To determine if AAV9-mediated mutant SOD1 reduction would slow diseaseprogression, a cohort of SOD1^(G37R) mice⁶ were injected intravenouslywith AAV9-SOD1-shRNA after disease onset (average age at treatment=215dversus median onset of 197d in treated animals; Log Rank Test p=0.46;FIG. 5a ). loxSOD1^(G37R) ALS mice, carrying a human mutant SOD1^(G37R)transgene flanked by lox p sites under its endogenous promoter, weremaintained in as previously described³⁷. A combination of AAV9-CB-GFP(n=9) and uninjected (n=12) littermates were used as controls.

Post hoc analysis showed no differences between GFP and uninjectedanimals, therefore the groups were compiled as “control” in FIG. 5.Animals were evaluated weekly for body weight and hind limb gripstrength and monitored until end-stage. AAV9-SOD1-shRNA treatment afterdisease onset significantly extended median survival by 86.5 days overcontrol animals (control, n=21, 392d; SOD1 shRNA, n=25, 478.5d; Log RankTest p<0.0001). Early disease duration, defined by the time from peakweight to 10% weight loss, was significantly slowed (control, 89d; SOD1shRNA treated mice, 162 days; Wilcoxon Signed Rank Test p<0.01; FIG. 5c). A continuing trend toward slowing of later disease (10% weight lossto end stage) was also seen (control, 63d; SOD1 shRNA treated mice, 81d;Wilcoxon Signed Rank Test p=0.1389; FIG. 5d ). Overall disease durationfollowing AAV9-SOD1-shRNA therapy rose to 239d after disease onsetversus 173d in control mice (Wilcoxon Signed Rank Test p<0.0001; FIG. 5e). Consistent with the slowed progression, AAV9 therapy maintained gripstrength relative to control SOD1 mutant animals (FIG. 5g ). The 86.5day extension in survival surpassed the 62 day extension seen intransgenic studies that used astrocyte-specific Cre expression toinactivate the mutant SOD1^(G37R) transgene⁸, presumably reflectingefficient AAV9 transduction of astrocytes after peripheral delivery andthe possible transduction of other cell types (especially microglia⁶)whose synthesis of mutant SOD1 accelerates disease progression.

Histological examination of end-stage SOD1^(G37R) treated animalsrevealed similar levels of intraspinal cell transduction in animalstreated with AAV9-SOD1-shRNA or AAV9-GFP (FIG. 6). GFP expression waspredominantly observed within motor neurons and astrocytes of bothgroups, and SOD1 expression was detectably decreased only in animalsthat received AAV9-SOD1-shRNA (FIGS. 6k,o ). Immunoblotting of wholespinal cord extracts from end stage SOD1^(G37R) mice revealed an 80%reduction in hSOD1 protein levels in AAV9-SOD1-shRNA treated animalscompared to controls (Figure S4).

Example 7 AAV9 Mediated Suppression of SOD1 in Non-Human Primates

To test whether SOD1 levels could be efficiently lowered using AAV9 inthe non-human primate spinal cord, AAV9 was injected intrathecally vialumbar puncture. This method was chosen over systemic delivery todecrease the amount of virus required and to minimize any effects fromreduction of SOD1 in peripheral tissues. One year old cynomolgusmacaques (Macaca fascicularis) with average body weight of 2 kg wereused for this study at the Mannheimer Foundation. Regular monitoring ofoverall health and body weight was performed prior and after theinjections to assess the welfare of the animals.

Sequencing of cDNA copied from mRNA isolated from African Green Monkey(COS cells) and the Cynomolgus macaque verified that the 130 shRNA had asingle base mismatch to either sequence (Figure S5). The 130 shRNAexpression cassette was inserted into a lentiviral vector which was thenused to transduce COS cells. Cos-7 cells were maintained in DMEM with10% FBS and 1% penicillin/streptomycin. Cells were infected with alentiviral vector expressing SOD1 shRNA 130 under the H1 promoter andRFP under CMV promoter. RNA was extracted from infected and non-infectedcells 72 hours post infection using an RNAeasy Kit (Qiagen). cDNA wasprepared using RT² First strand synthesis kit (SABiosciences). SOD1transcript levels were analyzed by qRT-PCR which revealed that themonkey SOD1 mRNA was reduced by ˜75% in 130 shRNA transduced cellscompared to mock transduced control cells (Figure S5).

The AAV9-SOD1-shRNA virus (1×10¹³ vg/kg) was infused along with contrastagent via lumbar puncture into the subarachnoid space of three malecynomolgus macaques and one control subject was injected withAAV9-CB-GFP (1×10¹³ vg/kg) (FIG. 7a ). Each intrathecal injection wasperformed by lumbar puncture into the subarachnoid space of the lumbarthecal sac. AAV9 was resuspended with omnipaque (iohexol), an iodinatedcompound routinely used in the clinical setting. Iohexol is used tovalidate successful subarachnoid space cannulation and was administeredat a dose of 100 mg/Kg. The subject was placed in the lateral decubitusposition and the posterior midline injection site at ˜L4/5 levelidentified (below the conus of the spinal cord). Under sterileconditions, a spinal needle with stylet was inserted and subarachnoidcannulation was confirmed with the flow of clear CSF from the needle. Inorder to decrease the pressure in the subarachnoid space, 0.8 ml of CSFwas drained, immediately followed by injection with a mixture containing0.7 mL iohexol (300 mg/ml formulation) mixed with 2.1 mL of virus (2.8ml total).

No side effects from the treatments were identified. Two weeks postinjection, the spinal cords were harvested for analysis of GFPexpression and SOD1 RNA levels. GFP expression was seen broadly inneuronal and astrocytic cells throughout the grey and white matter ofthe lumbar spinal cord, the area closest to the site of injection (FIGS.7b-e ). Immunoblotting of extracts of lumbar spinal cord revealed 87%reduction in monkey SOD1 protein levels (FIGS. 7f,g ). Laser capturemicrodissection was then used to isolate total RNA from motor neurons aswell as from glia in the nearby neuropil. Analysis by quantitativeRT-PCR using primers specific for monkey SOD1 (and normalized to actin)confirmed a 95±3% knockdown in the motor neuron pool and a 66±9%knockdown in the neuropil pool when compared to samples from a controlanimal (FIG. 7h ).

Next we examined the level of cell transduction throughout the spinalcord including cervical, thoracic and lumbar segments. GFP was found tobe expressed broadly within all sections analyzed (FIGS. 8a-c ). Motorneuron counts revealed a caudal to rostral gradient in celltransduction, with the cervical region showing more than 50% ofGFP/Chat+ motor neurons, increasing to 65% in the thoracic region andreaching a remarkable 80% in the lumbar region (FIG. 8d ). In order todetermine the overall level of SOD1 knockdown achieved with thistransduction pattern, qRT-PCR for SOD1 was performed on whole sectionhomogenates from cervical, thoracic and lumbar cord segments. Theresults confirmed robust SOD1 reduction at all three spinal cord levels,ranging from a 60% decrease in the cervical segment, a 70% decrease inthe thoracic region and an 88% decrease in the lumbar region (FIG. 8e ),consistent with the proportion of cells transduced in each region.

DISCUSSION

The examples above show that intravenous administration ofAAV9-SOD1-shRNA is safe and well tolerated in wild type mice, with theabsence of adverse effects after long-term assessment. Thiss approachhave achieved one of the longest extensions in survival ever reported inthe rapidly progressive SOD1^(G93A) mouse model of ALS (increasingsurvival by 39% when treatment is initiated at birth). Even moreencouraging, markedly slowed disease progression is seen even when AAV9therapy to reduce mutant SOD1 synthesis is applied after disease onsetin SOD1^(G37R) mice, thereby significantly extending survival. Thus, thevascular delivery paradigm in mice represents a proof of concept thatmutant SOD1 knockdown after disease onset can be beneficial in bothrapid and more slowly progressive models of ALS at clinically relevantpoints in disease. Together, these data show that robust targeting andsuppression of SOD1 levels via AAV9-mediated delivery of shRNA iseffective in slowing disease progression in mouse models of ALS,critically even when treatment is initiated after onset.

Multiple recent studies have brought forward the hypothesis thatwild-type SOD1 may contribute through misfolding to the pathogenicmechanism(s) that underlie sporadic ALS through a pathway similar tothat triggered by mutant SOD1^(14,30-32). Included in this body ofevidence is our own demonstration that astrocytes produced from sporadicALS patients are toxic to co-cultured motor neurons and that toxicity isalleviated by siRNA-mediated reduction in wild type SOD1³⁰. Thisevidence creates the potential that a proportion of sporadic ALSpatients could also benefit from an AAV9-mediated SOD1 reductionapproach that we have demonstrated to be effective in slowing diseaseprogression in mice that develop fatal, ALS-like disease from expressingALS-causing mutations in SOD1.

Finally, for translation of an AAV9-mediated suppression of SOD1synthesis to the human setting, we have determined that infusiondirectly into the CSF at the lumbar level in a non-human primate producesubstantial SOD1 reduction by targeting both motor neurons andnon-neuronal cells. This outcome provides strong support for extendingthese efforts to an adult human by direct injection into CSF, aspreviously proposed^(33,34), so as to 1) limit the cost of viralproduction, 2) reduce the possibility that chronic suppression of SOD1in the periphery may have deleterious consequences, and 3) reduce viralexposure to the peripheral immune system³³. These data strongly indicateAAV9-SOD1-shRNA as a treatment for ALS.

Techniques/Methods Used in Examples 1-7

Perfusion and Tissue Processing.

Control and treated SOD1^(G93A) mice were sacrificed at either 21 dayspost injection or at endstage for immunohistochemical analysis. Animalswere anesthetized with xylazene/ketamine cocktail, transcardiallyperfused with 0.9% saline, followed by 4% paraformaldehyde. Spinal cordswere harvested, cut into blocks of tissue 5-6 mm in length, and then cutinto 40 μm thick transverse sections on a vibratome (Leica, Bannockburn,Ill.). Serial sections were kept in a 96-well plate that contained 4%paraformaldehyde and were stored at 4° C. End stage loxSOD1^(G37R) micewere anesthetized using isoflurane and perfused with 4%paraformaldehyde. Spinal cord segments, including cervical, thoracic andlumbar segments were dissected. Following cryoprotection with 20%sucrose/4% paraformaldehyde overnight, spinal cords were frozen inisopentane at −65° C., and serial 30 μm coronal sections were collectedfree floating using sliding microtome.

For safety studies, P1, P21 treated and control wild type mice weresacrificed at 180 days of age. Animals were anesthetized usingxylazene/ketamine cocktail and perfused with 0.9% saline. Differenttissues were removed and stored in 10% buffered formalin. These tissueswere further processed, blocked and mounted for hematoxilin & eosinstaining by the Nationwide Children's Hospital Morphology Core.

Cynomolgus monkeys injected with virus were euthanized 2 weeks postinjection. Animals were anesthetized with sodium pentobarbital at thedose of 80-100 mg/kg intravenously and perfused with saline solution.Brain and spinal cord dissection were performed immediately and tissueswere processed either for nucleic acid isolation (snap frozen) orpost-fixed in 4% paraformaldehyde and subsequently cryoprotected with30% sucrose and frozen in isopentane at −65° C. 12 μm coronal sectionswere collected from lumbar cord using a cryostat for free floatingimmunostaining.

Immunohistochemistry. Mouse spinal cords were stained as floatingsections. Tissues were washed three-times for 10 minutes each in TBS,then blocked in a solution containing 10% donkey serum, 1% Triton X-100and 1% penicillin/streptomycin for two hours at room temperature. Allthe antibodies were diluted with the blocking solution. Primaryantibodies used were as follows: rabbit anti-GFP (1:400, Invitrogen,Carlsbad, Calif.), rabbit anti-SOD1 (1:200, Cell signaling, Danvers,Mass.), goat anti-ChAT (1:50 Millipore, Billerica, Mass.), mouseanti-GFAP (1:200, Millipore, Billerica, Mass.), chicken anti GFAP(1:400, Abcam, Cambridge, Mass.), and rabbit anti-Ibal (1:400, Wako,Richmond Va.). Tissues were incubated in primary antibody at 4° C. for48-72 hours then washed three times with TBS. After washing, tissueswere incubated for 2 hours at room temperature in the appropriate FITC-,Cy3-, or Cy5-conjugated secondary antibodies (1:200 JacksonImmunoresearch, Westgrove, Pa.) and DAPI (1:1000, Invitrogen, Carlsbad,Calif.). Tissues were then washed three times with TBS, mounted ontoslides then coverslipped with PVA-DABCO. All images were captured on aZeiss-laser-scanning confocal microscope.

For DAB staining, monkey spinal cord sections were washed three times inTBS, blocked for 2 h at RT in 10% donkey serum and 1% Triton X-100.Sections were then incubated overnight at 4° C. with rabbit anti-GFPprimary antibody (1:1000 Invitrogen, Carlsbad, Calif.) diluted inblocking buffer. The following day, tissues were washed with TBS 3times, incubated with biotinylated secondary antibody anti-rabbit (1:200Jackson Immunoresearch, Westgrove, Pa.) in blocking buffer for 30 min atRT, washed 3 times in TBS and incubated for 30 min at RT with ABC(Vector, Burlingame, Calif.). Sections were then washed for 3 times inTBS and incubated for 2 min with DAB solution at RT and washed withdistilled water. These were then mounted onto slides and covered withcoverslips in mounting medium. All images were captured with the ZeissAxioscope.

Motor Neuron and Astrocyte Quantification.

For MN quantification, serial 40 μm thick lumbar spinal cord sections,each separated by 480 μm, were labeled as described for GFP and ChATexpression. Stained sections were serially mounted on slides fromrostral to caudal, then coverslipped. Sections were evaluated usingconfocal microscopy (Zeiss) with a 40× objective and simultaneous FITCand Cy3 filters. The total number of ChAT positive cells found in theventral horns with defined soma was tallied by careful examinationthrough the entire z-extent of the section. GFP labeled cells werequantified in the same manner, while checking for co-localization withChAT. For astrocyte quantification, as with MNs, serial sections werestained for GFP, GFAP and then mounted. Using confocal microscopy with a63× objective and simultaneous FITC and Cy5 filters, random fields inthe ventral horns of lumbar spinal cord sections from tail vein injectedanimals were selected. The total numbers of GFP and GFAP positive cellswere counted from a minimum of at least 24-fields per animal whilefocusing through the entire z extent of the section. Spinal cordsections of 3 animals per group were examined for MN and astrocytequantification.

Immunoblot Analysis.

Spinal cords were harvested from P1, P21 injected and controlSOD1^(G93A) mice 21 days post injection and from treated and controlmonkeys 2 weeks post injection of AAV9-SOD1-shRNA. Spinal cords werehomogenized and protein lysates were prepared using T-Per (Pierce) withprotease inhibitor cocktail. Samples were resolved on SDS-PAGE accordingto manufacturer's instructions. Primary antibodies used were rabbitanti-SOD1 (1:750, Cell signaling, Danvers, Mass.) mouse anti-SOD1(1:750, Millipore, Billerica, Mass.), rabbit anti-SOD1 (1:1000, Abcam,Cambridge, Mass.), rabbit anti-Actin (1:1000, Abcam, Cambridge, Mass.)and mouse anti-GAPDH (1:1000, Millipore, Billerica, Mass.). Secondaryantibodies used were anti-rabbit HRP (1:10000-1:50000) and anti-mouseHRP (1:10000). Densitometric analysis was performed using Image Jsoftware.

Laser Capture Microdissection.

12 □m lumbar spinal cord frozen sections were collected onto PENmembrane slides (Zeiss, Munich, Germany) and stained with 1% Cresylviolet (Sigma, St. Louis, Mo.) in methanol. Sections were air dried andstored at −80° C. After thawing, motor neurons were collected within 30min from staining using the laser capture microdissector PALM Robo3Zeiss) using the following settings: Cut energy: 48, LPC energy: 20, Cutfocus: 80/81, LPC focus: 1, Position speed: 100, Cut speed: 50. About500 MNs were collected per animal. Non-neuronal cells from the ventralhorn were collected from the same sections after collecting the motorneurons.

qRT-PCR.

RNA from laser captured cells or whole spinal cord sections from thecervical, thoracic and lumbar segments was isolated using the RNaqueousMicro Kit (Ambion, Grand Island, N.Y.) according to manufacturer'sinstructions. RNA was then reverse-transcribed into cDNA using the RT²HT First Strand Kit (SABiosciences, Valencia, Calif.). 12.5 ng RNA wereused in each Q-PCR reaction using SyBR Green (Invitrogen, Carlsbad,Calif.) to establish the relative quantity of endogenous monkey SOD1transcript in animals who had received the AAV9-SOD1-shRNA compared toanimals who had received only AAV9-GFP. Each sample was run intriplicate and relative concentration calculated using the ddCt valuesnormalized to endogenous actin transcript.

Behavior and Survival Analysis. Treated and control SOD1^(G93A) micewere monitored for changes in body mass twice a week. loxSOD1^(G37R)mice were weighed on a weekly basis. Motor coordination was recordedusing a rotarod instrument (Columbus Instruments, Columbus, Ohio). Eachweekly session consisted of three trials on the accelerating rotarodbeginning at 5 rpm/min. The time each mouse remained on the rod wasregistered. Both SOD1^(G93A) and loxSOD1^(G37R) mice were subjected toweekly assessment of hindlimb grip strength using a grip strength meter(Columbus Instruments, Columbus, Ohio). Each weekly session consisted of3 (SOD1^(G93A) mice) or 5 (loxSOD1^(G37R) mice) tests per animal.Survival analysis was performed using Kaplan-Meier survival analysis.End stage was defined as an artificial death point when animals could nolonger “right” themselves within 30 sec after being placed on its back.Onset and disease progression were determined from retrospectiveanalysis of the data. Disease onset is defined as the age at which theanimal reached its peak weight. Disease duration is defined as the timeperiod between disease onset and end stage. Early disease duration isthe period between peak weight and loss of 10% of body weight while latedisease duration is defined as the period between 10% loss of bodyweight until disease end stage. Due to shorter life span of SOD1^(G93A)animals, we did not assess the distinction between the early and lateprogression.

For toxicity analysis following injection at P1 or P21, treated andcontrol WT mice were subjected to behavioral analysis starting at −30days of age and monitored up to 6 months. Body mass was recorded weeklywhile rotarod performance and hindlimb grip strength were recordedbiweekly.

Hematology and Serum Studies.

Blood samples were collected in (K2) EDTA microtainer tubes (BD) fromtreated and control WT mice at 150 days of age by mandibular veinpuncture. The same animals were bled at 180 days of age and blood wascollected in serum separator microtainer tubes. The blood was allowed toclot for an hour and was then centrifuged at 10,000 rpm for 5 minutes.The clear upper phase (serum) was collected and frozen at −80° C.Hematological and serum analysis were conducted by Ani Lytics Inc,Gaithersburg, Md.

Statistical analysis. All statistical tests were performed using theGraphPad Prism (San Diego, Calif.) software package. Kaplan Meiersurvival analyses were analyzed by the Log Rank Test. Comparisons ofmedian disease durations and survival times were analyzed by theWilcoxon Signed Rank Test.

Example 8 Development of a Clinical SOD1 shRNA Construct

The AAV SOD1 shRNA vector described in Example 2 carries shRNA againsthuman SOD1 sequence under the H1 promoter (FIG. 9a ). The same vectoralso contains a GFP expression cassette which expresses GFP under a CBApromoter. The other regulatory elements present in this cassette includeCMV enhancer, SV40 intron and bGH PolyA terminator sequence. We showherein that AAV9 SOD1 shRNA administration results in efficient SOD1downregulation along with robust expression of GFP in vitro as well asin vivo. No significant alterations were observed after the long termassessment of wild-type mice administered with AAV9 SOD1 shRNA. Theseresults suggested that there are no evident off-target effects due tothe long-term expression of SOD1 shRNA as well as overexpression of GFP.Although we did not find GFP toxicity in our mice, several reports haveshown the adverse effects of GFP overexpression in vitro and in vivo.Therefore, to eliminate the possibility of GFP toxicity altogether, theSOD1 shRNA construct of Example 2 was modified by replacing the GFPexpression cassette with a non-coding stuffer sequence while maintainingthe size of the total DNA construct flanked by the ITRs (FIG. 9b ). Thisis important as the distance between the two ITR sequences greatlyaffects the packaging capacity of the flanked construct into AAV9capsids[321-324].

To date, none of the FDA approved stuffer sequences are readilyavailable. There are, however, several plasmid backbones that areapproved by FDA for the human administration. Small DNA fragments werepicked from these plasmids which do not correspond to any essential DNAsequences necessary for selection and replication of the plasmid or theelements of the transcriptional units. The plasmid backbones are listedin Table 1. The DNA elements from different plasmids were arranged intandem to generate a complete, 1607 bp stuffer sequence (SEQ ID NO: 22).Finally, a DNA construct containing the SOD1 shRNA expression cassette,followed by the stuffer sequence was synthesized from Genscript.

TABLE 1 Plasmid ClinicalTrials.gov Backbone Condition Intervention PhaseIdentifier pVax1 Early Stage Non-Small Recombinant DNA- 1 NCT00062907Cell Lung Cancer pVAX/L523S pCDNA3 Chronic Hepatitis B DNA vaccine 1, 2NCT00536627 pCMVS2.S pUCMV3 Stage III Ovarian pUMVC3-hIGFBP-2 1NCT01322802 Epithelial Cancer multi-epitope plasmid Stage III OvarianDNA vaccine Germ Cell Tumor Stage IV Ovarian Epithelial Cancer Stage IVOvarian Germ Cell Tumor pBK-CMV Prostate Cancer NY-ESO-1 plasmid 1NCT00199849 Bladder Cancer DNA Cancer Vaccine Non-Small Cell Lung CancerEsophageal Cancer Sarcoma pGA2 HIV Infections pGA2/JS2 Plasmid DNA 1NCT00043511 Vaccine

Clinical SOD1 shRNA construct has shRNA against human SOD1 under H1promoter which is followed by the non-coding stuffer sequence. Thisconstruct is designed in such a way that multiple shRNA expressioncassettes can be added to the final vector by simultaneous removal ofthe stuffer sequence. Restriction endonuclease sites have been added tothe stuffer sequence so that a part of the stuffer can be removed whenanother shRNA expression cassette is added (FIG. 10). This simultaneousremoval and addition of DNA sequences would help maintaining the optimalsize of the whole construct between the ITRs (˜2.0 kb) to achieveefficient packaging.

Clinical SOD1 shRNA construct from Genscript was cloned into pJet1.2shuttle vector via EcoRV. This parental clone was screened using variousrestriction endonucleases designed within the construct to confirm thecorrect clone. Kpn1/Sph1 double digestion of pJet SOD1 shRNA confirmedthe presence of the complete construct (2023 bp) while Xba1 digestionconfirmed the presence of SOD1 shRNA expression cassette (414 bp) andthe stuffer element, along with pJet backbone (3000 bp). EcoRV/Pme1double digestion also revealed the presence of stuffer element.

Example 9 Clinical SOD1 shRNA Efficiently Reduces Human SOD1 ProteinLevels In Vitro

To determine the efficacy of the de novo synthesized SOD1 shRNAconstruct to downregulate SOD1 levels, HEK293 cells were transfectedwith pJet SOD1 shRNA plasmid using Calcium Phosphate method. AAV SOD1shRNA plasmid was used as a positive control. Immunofluorescenceanalysis of HEK293 cells, 72 hrs post transfection revealed the lack ofnative GFP fluorescence from pJet SOD1 shRNA transfected cells ascompared to AAV9 SOD1 shRNA transfected cells. Immunoblot analysis ofcell lysates from these cells further confirmed the successfulreplacement of GFP from pJet SOD1 shRNA plasmid. Importantly, pJet SOD1shRNA resulted in efficient downregulation of SOD1 protein levels(>50%), similar to AAV SOD1 shRNA plasmid. See FIG. 11.

Example 10 Generation of Clinical AAV SOD1 shRNA

Clinical SOD1 shRNA construct was further cloned into an AAV.CB.MCSvector using Kpn1/Sph1 sites to generate clinical AAV SOD1 shRNA plasmid(FIG. 12). AAV.CB.MCS was generated from AAV.CB.GFP plasmid obtainedfrom merion Scientific by replacing GFP with a multiple cloning site(MCS). Cloning of clinical SOD1 shRNA construct at Kpn1/Sph1 sites putsit between the two AAV2 ITRs which facilitates the packaging of theconstruct in AAV9 viral capsids. See FIG. 12.

Clinical AAV SOD1 shRNA plasmid was screened with restrictionendonucleases to confirm the presence of SOD1 shRNA expression cassette(Xba1 digest), stuffer sequence (EcoRV/Pme1 double digest) and alsointact ITR sequences (Sma1 digest).

Example 11 Clinical AAV SOD1 shRNA Efficiently Reduces Human SOD1Protein Levels In Vitro

Clinical AAV SOD1 shRNA plasmid was transfected in HEK293 cells todetermine its knockdown efficiency. Similar to the pJet SOD1 shRNAplasmid, clinical AAV SOD1 shRNA transfected cells were devoid of anyGFP expression as evident by immunofluorescence (FIG. 13a-f ) andimmunoblot assay (FIG. 13g ). More importantly, clinical AAV SOD1 shRNAefficiently reduced human SOD1 protein levels in HEK293 cells by morethan 50% (FIG. 13g,h ). Altogether, these results confirmed thesuccessful generation of clinical AAV SOD1 shRNA vector with functionalSOD1 shRNA expression cassette and complete removal of the transgeneexpression cassette.

DOCUMENTS REFERENCED

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We claim:
 1. A recombinant adeno-associated virus comprising thesuperoxide dismutase 1 (SOD1) shRNA-encoding DNA: (SEQ ID NO: 1)GCATCATCAATTTCGAGCAGAAGGAA, (SEQ ID NO: 2) GAAGCATTAAAGGACTGACTGAA,(SEQ ID NO: 3) CTGACTGAAGGCCTGCATGGATT, (SEQ ID NO: 4)CATGGATTCCATGTTCATGA, (SEQ ID NO: 5) GCATGGATTCCATGTTCATGA,(SEQ ID NO: 6) GGTCTGGCCTATAAAGTAGTC, (SEQ ID NO: 7)GGGCATCATCAATTTCGAGCA, (SEQ ID NO: 8) GCATCATCAATTTCGAGCAGA,(SEQ ID NO: 9) GCCTGCATGGATTCCATGTTC, (SEQ ID NO: 10)GGAGGTCTGGCCTATAAAGTA, (SEQ ID NO: 11) GATTCCATGTTCATGAGTTTG,(SEQ ID NO: 12) GGAGATAATACAGCAGGCTGT, (SEQ ID NO: 13)GCTTTAAAGTACCTGTAGTGA, (SEQ ID NO: 14) GCATTAAAGGACTGACTGAAG,(SEQ ID NO: 1) GCATCATCAATTTCGAGCAGAAGGAA, (SEQ ID NO: 2)GAAGCATTAAAGGACTGACTGAA, (SEQ ID NO: 3) CTGACTGAAGGCCTGCATGGATT,(SEQ ID NO: 4) CATGGATTCCATGTTCATGA, (SEQ ID NO: 5)GCATGGATTCCATGTTCATGA, (SEQ ID NO: 6) GGTCTGGCCTATAAAGTAGTC,(SEQ ID NO: 7) GGGCATCATCAATTTCGAGCA, (SEQ ID NO: 8)GCATCATCAATTTCGAGCAGA, (SEQ ID NO: 9) GCCTGCATGGATTCCATGTTC,(SEQ ID NO: 10) GGAGGTCTGGCCTATAAAGTA, (SEQ ID NO: 11)GATTCCATGTTCATGAGTTTG, (SEQ ID NO: 12) GGAGATAATACAGCAGGCTGT,(SEQ ID NO: 13) GCTTTAAAGTACCTGTAGTGA, (SEQ ID NO: 14)GCATTAAAGGACTGACTGAAG, (SEQ ID NO: 15) TCATCAATTTCGAGCAGAA,(SEQ ID NO: 16) TCGAGCAGAAGGAAAGTAA, (SEQ ID NO: 17)GCCTGCATGGATTCCATGT, (SEQ ID NO: 18) TCACTCTCAGGAGACCATT,  or(SEQ ID NO: 19) GCTTTAAAGTACCTGTAGT,

wherein the recombinant adeno-associated virus genome lacks rep and capgenes.
 2. A composition comprising the recombinant adeno-associatedvirus of claim
 1. 3. A method of inhibiting expression of mutant SOD1 ina cell comprising contacting the cell with a recombinantadeno-associated virus of claim 1 or the composition of claim
 2. 4. Amethod of delivering the SOD1 shRNA-encoding DNAGCATCATCAATTTCGAGCAGAAGGAA (SEQ ID NO:1) to a subject in need thereof,comprising administering to the subject a recombinant adeno-associatedvirus comprising the SOD1 shRNA-encoding DNA GCATCATCAATTTCGAGCAGAAGGAA(SEQ ID NO:1), wherein the recombinant adeno-associated virus genomelacks rep and cap genes.
 5. A method of delivering the SOD1shRNA-encoding DNA GAAGCATTAAAGGACTGACTGAA (SEQ ID NO:2) to a subject inneed thereof, comprising administering to the subject a recombinantadeno-associated virus comprising the SOD1 shRNA-encoding DNAGAAGCATTAAAGGACTGACTGAA (SEQ ID NO:2), wherein the recombinantadeno-associated virus genome lacks rep and cap genes.
 6. A method ofdelivering the SOD1 shRNA-encoding DNA CTGACTGAAGGCCTGCATGGATT (SEQ IDNO:3) to a subject in need thereof, comprising administering to thesubject a recombinant adeno-associated virus comprising the SOD1shRNA-encoding DNA CTGACTGAAGGCCTGCATGGATT (SEQ ID NO:3), wherein therecombinant adeno-associated virus genome lacks rep and cap genes.
 7. Amethod of delivering the SOD1 shRNA-encoding DNA CATGGATTCCATGTTCATGA(SEQ ID NO:4) to a subject in need thereof, comprising administering tothe subject a recombinant adeno-associated virus comprising the SOD1shRNA-encoding DNA CATGGATTCCATGTTCATGA (SEQ ID NO:4), wherein therecombinant adeno-associated virus genome lacks rep and cap genes.
 8. Amethod of treating amyotrophic lateral sclerosis (ALS) comprisingadministering a recombinant adeno-associated virus comprising the SOD1shRNA-encoding DNA GCATCATCAATTTCGAGCAGAAGGAA (SEQ ID NO:1), wherein therecombinant adeno-associated virus genome lacks rep and cap genes.
 9. Amethod of treating ALS comprising administering a recombinantadeno-associated virus comprising the SOD1 shRNA-encoding DNAGAAGCATTAAAGGACTGACTGAA (SEQ ID NO:2), wherein the recombinantadeno-associated virus lacks rep and cap genes.
 10. A method of treatingALS comprising administering a recombinant adeno-associated viruscomprising the SOD1 shRNA-encoding DNA CTGACTGAAGGCCTGCATGGATT (SEQ IDNO:3), wherein the recombinant adeno-associated virus lacks rep and capgenes.
 11. A method of treating ALS comprising administering arecombinant adeno-associated virus comprising the SOD1 shRNA-encodingDNA CATGGATTCCATGTTCATGA (SEQ ID NO:4), wherein the recombinantadeno-associated virus lacks rep and cap genes.